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CHAPTER 17
Red blood cell enzyme defects
Joan-Luis Vives Corrons
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CHAPTER 17 • Red blood cell enzyme defects
1. Introduction
The maturation process of erythroblasts involves the disappearance of virtually all
the metabolic pathways specific to any other cell in the body, leaving only the
components essential for maintaining the supply of energy and the defence against
oxidising agents. The mature red blood cell (RBC), however, is optimally adapted
to perform its most important function during its estimated 120-day lifespan in the
circulation: the binding, transport and delivery of oxygen to all tissues. For this,
the RBC requires three essential metabolic pathways:
• Anaerobic glycolysis, which is the only source of energy (ATP production) for
maintenance of cell structure and function
• Anti-oxidant pathways necessary for the protection of RBC proteins against
oxidation, through the synthesis of glutathione (GSH), and of haemoglobin
against iron oxidation through the maintenance of iron in its functional, reduced,
ferrous state (cytochrome b5 reductase)
• Nucleotide metabolism for the maintenance of the purine and pyrimidine
nucleotides.
Moreover, erythrocytes possess a unique glycolytic bypass for the production of 2,3bisphosphoglycerate (2,3-BPG) a crucial metabolite in the regulation of haemoglobin
affinity for oxygen. RBC enzymopathies have been described in all these metabolic
pathways and almost all are associated with chronic haemolytic anaemia (CHA), with
the exception of the enzymopathies of the pentose phosphate pathway and
glutathione metabolism, which are associated with acute haemolytic crises only after
exposure to oxidant substances.
Some of the most rare enzyme defects are also expressed in other tissues. In
general, however, the deficiency is more pronounced in RBCs, when compared with
other cells, because of the long lifespan of the mature erythrocyte after the loss
of protein synthesis. In general, these enzymopathies are associated, in addition
to haemolytic anaemia (acute or chronic), with systemic (non-haematological)
manifestations such as neuropathy (with or without mental retardation), muscular
disease, recurrent infections and metabolic acidosis.
By far the majority of red cell enzymopathies are hereditary in nature, although
acquired deficiencies have also been described, mainly in malignant haematological
disorders (1).
Since the discovery of glucose 6-phosphate dehydrogenase (G6PD) deficiency in 1956,
and pyruvate kinase (PK) deficiency in 1961, erythroenzymopathies associated with
hereditary haemolytic anaemia have been extensively investigated (2-4). The mode
of inheritance is autosomal recessive for almost all of erythroenzymopathies, but
autosomal dominant for ADA overproduction and X-linked for G6PD and PGK
deficiencies. In general, the genetic mutations that cause RBC enzyme defects are
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associated with three different clinical phenotypes: a) haemolytic syndrome
associated with either chronic anaemia (CHA) or acute haemolytic crises, b)
permanent cyanosis with methaemoglobinaemia and c) increased RBC mass or
erythrocytosis.
2. Erythroenzymopathies associated with hereditary haemolytic anaemia
Erythroenzymopathies associated with CHA show common findings and generally
differ greatly from case to case, even in one enzyme deficiency or within the same
family. The degree of haemolysis is dependent on the severity of enzyme deficiency,
a consequence of the properties of the mutant enzyme with regard to functional
abnormalities, molecular instability, or both. In addition to the anaemia, other
clinical findings are jaundice, due to increased indirect-reacting bilirubin,
reticulocytosis, and splenomegaly. RBC morphology usually shows anisocytosis and
polychromasia without specific changes, with the exception of echinocytosis,
eccentrocytosis and basophilic stippling, which are frequently seen in PK, G6PD
and P5’N deficiencies, respectively. The morphological abnormalities such as
spherocytosis and elliptocytosis characteristic of some RBC membrane disorders,
are not seen in erythroenzymopathies. This is the reason why erythroenzymopathies
belong to the so-called hereditary non-spherocytic haemolytic anaemias (HNSHA).
In most cases of G6PD deficiency, acute haemolytic anaemia (AHA) is caused either
by the ingestion of oxidant drugs, such as salicylic acid or antimalarials (primaquine)
or by infection. In some cases, haemolysis is seen after ingestion of fava beans
(favism). Anaemia and/or neonatal jaundice are also common features. Although
glutathione reductase (GR) deficiency would cause the same symptoms, and is
associated with favism, it is extremely rare.
Diagnosis: The most frequent human enzymopathy, G6PD deficiency, can be diagnosed
by a simple screening test (5). However, when the patient suffers from a longstanding CHA either PK deficiency or another glycolytic enzymopathy, or an unstable
haemoglobin should be suspected. The haemoglobin heat stability test is a simple and
reliable screening test to detect unstable haemoglobins. However, a direct RBC enzyme
activity assay is necessary to confirm the diagnosis of RBC enzyme deficiencies (6).
In hereditary PK deficiency it is important to remove leukocytes from RBC suspension
before making the red cell haemolysate because leukocytes possess 300 times more
PK activity than RBCs. In addition, leukocytes have a different isoenzyme, which has
normal activity in PK deficient cases. Currently, DNA diagnosis has become an available
procedure for the study of almost all erythroenzymopathies. However, it is still not simple
because there are often several or many mutations even in one enzyme deficiency. If
the mutation(s) in a particular family is (are) already known, DNA diagnosis is
relatively easy, and prenatal diagnosis is possible. Normal genomic DNA or cDNA for
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CHAPTER 17 • Red blood cell enzyme defects
most of the enzymes causing hereditary haemolytic anaemia has been isolated allowing
a rapid advancing in the understanding of the molecular basis of RBC enzyme defects
associated with hereditary haemolytic anaemia.
PK and class I G6PD deficiencies comprise the two most common erythroenzymopathies
associated with CHA followed by glucose-phosphate isomerase (GPI) and pyrimidine
5’- nucleotidase deficiencies. Very rare enzymopathies include the deficiencies of
hexokinase (HK) adenylate kinase (AK), phosphofructokinase (PFK), phosphoglycerate
kinase (PGK), triose-phosphate isomerase (TPI), glutathione peroxidase (GSH-Px),
glutathione reductase (GR), glutathione synthetase (GS), gamma-glutamylcysteine
synthetase (GGCS) and the overproduction of adenosine deaminase (ADA). Considered
together, all these enzymopathies account for less than 10 percent of all the patients
with enzyme deficiency associated with CHA. Moreover in some cases, the CHA is
associated with severe clinical manifestations like neuropathy (TPI, PGK, GGCS, GS and
AK) and/or myopathy (PFK, PGK, AK) or frequent infections (TPI). Almost all these
enzymes are located in the two main metabolic pathways of RBC: anaerobic glycolysis
and anti-oxidative metabolism (Figure 1).
Figure 1: RBC metabolism. Anaerobic glycolysis and antioxidant pathways
GSH
NADP
Glucose
CO2
ATP
ADP
HK
G6P
6PG
G6PD
GPI
Glutathione
peroxidase
Glutathione
reductase
NADPH
GSSG
6PGD
Ru5P
F6P
ATP
ADP
PFK
H2O
2H +
GSH
GSH
ANTIOXIDANT METABOLISM
FDP
Aldolase
H2O2
ADP
ATP
Glycine
Glutathione-synthetase
γ-glutamyl-cysteine
DHAP
G3P
G3PD
ANAEROBIC
GLYCOLYSIS
Pi
NAD
NADH
1,3-BPG
Mutase
2,3-BPG
Phosphatase
3-PG
Mutase
GCS
ADP
ATP
TPI
Cysteine
Glutamic acid
PGK
ADP
ATP
ATP ADP
2-PG
PEP
Enolase
PK
NADH
NAD
Pyruvate
Lactate
Lactate dehydrogenase
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General aspects of treatment and prevention: There is no treatment for hereditary RBC
enzyme deficiencies. Non-specific supportive measures such as red cell transfusions
provide the only currently accepted approach. Splenectomy is not curative but is
frequently of value, especially in infants and young children with severe disease.
Haemoglobin concentration often increases by 1-3 g/dL, reducing or even eliminating
transfusion requirements. Phototherapy is recommended in case of neonatal
hyperbilirubinaemia, but exchange transfusion may become necessary in severe
cases. G6PD-deficient patients should be advised to avoid potentially haemolytic drugs
or, in case of indispensable drugs, to ensure that they are used in sub-haemolytic doses.
2.1 Glucose-6-phosphate dehydrogenase (G6PD) deficiency
G6PD catalyses the first step in the hexose monophosphate shunt which is necessary
for producing NADPH. NADPH in turn is required for the maintenance of reduced
glutathione (GSH), a tripeptide that protects the RBC from oxidative damage
(Figure 1). G6PD is distributed in all cells and the active enzyme is a monomer of
515 aminoacids, with a molecular weight of about 59 kDa. The enzyme is active as
a tetramer or dimmer, depending on pH (7).
2.1.1 Clinical manifestations
G6PD deficiency (OMIM 305 900) is the most common known enzymopathy and it is
estimated to affect 400 million people worldwide. G6PD deficiency is X-linked and caused
by different mutations in the G6PD gene, resulting in protein variants with different levels
of enzyme activity that are associated with a wide range of biochemical and clinical
phenotypes. Major clinical manifestations are AHA after ingestion of drug or fava
beansand/or neonatal jaundice. A very small proportion of G6PD-deficient individuals
have a CHA and this has been defined by World Health Organisation (WHO) classification
as Class I G6PD-deficiency (see below). Favism presents as an acute haemolytic crisis,
usually after 24 to 72 h of ingestion of the beans (or contact with fava bean products)
with severe anaenia (sometimes requiring blood transfusion) and haemoglobinuria
(dark urine), which is more severe than that caused by drugs or infection, although
bilirubin concentration is slightly lower. In the acute stages of favism a number of
characteristic changes in RBC morphology are frequently observed, including
eccentrocytosis (Figure 2A) or bite-cells (Figure 2B). The anaemia worsens until days
7–8 and after drug cessation, haemoglobin concentrations recover after approximately
12 days. Heinz bodies (denatured haemoglobin precipitates) in peripheral red blood
cells, detected by methyl violet staining, are a typical finding (Figure 3). Favism can
develop after ingestion of dried or frozen beans, but is particularly likely to occur after
eating fresh beans; the disorder is most frequent in the period when beans are harvested.
Although favism was first noted to be common in Mediterranean countries, it was
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CHAPTER 17 • Red blood cell enzyme defects
Figure 2: MGG stained blood smear from a patient with G6PD deficiency and
acute haemolytic crisis of favism
A
B
A. Several erythrocytes with a ragged appearing, poorly haemoglobinised fringe of cytoplasm along one
side of the cell can be seen. This appearance (eccentrocytes or hemi-ghosts) results from an apposition
and adherence of opposing inner surfaces of the cell membrane with a variable exclusion of
haemoglobinised cytoplasm to an "eccentric" location in the cell. B. Several bite-cells or red blood cells
(RBCs) from which, one or more semicircular portions have been removed from the cell margin.
subsequently also observed in other geographical areas such as Middle East, the Far
East, and North Africa. Most patients who develop favism have the Mediterranean variant
of G6PD deficiency but favism is also very common with the G6PD A-variant in
populations where the growth and consumption of fava beans is widespread.
Drugs capable of producing an acute
haemolytic crisis in G6PD deficiency can
Figure 3: Methyl violet stained RBCs
be classified into two main groups: a)
from a patient with G6PD deficiency
drugs that have been shown to be
showing a characteristic inclusion of
capable of producing clinically significant
denatured haemoglobin (Heinz bodies)
haemolytic anaemia in doses that are
normally used (Table 1) and b) drugs that
have been shown to have a minor effect
on red cell survival when given in large
doses, such as aspirin, but which can be
given safely to most patients with G6PD
deficiency (Table 2).
Patients with CHA due to G6PD deficiency
have, in general, a history of severe
neonatal jaundice, chronic anaemia
exacerbated by oxidative stress, blood
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Table 1: Drugs and chemicals which should be avoided by all persons with G6PD
deficiency
Acetanilid
Phenazopyridine (Pyridium)
Diaminodiphenyl sulfone
Phenylhydrazine
Furazolidone (Furoxone)
Primaquine
Glibenclamide
Sulfacetamide
Henna (Lawsone)
Sulfanilamide
Isobutyl nitrite
Sulfapyridine
Methylene Blue
Thiazolesulfone
Naphthalene
Trinitrotoluene (TNT)
Niridazole (Ambilhar)
Urate oxidase
Nitrofurantoin (Furadantin)
Beutler E. Glucose-6-phosphate dehydrogenase deficiency: A historical perspective. Blood 2008; 111: 16-24
Table 2: Drugs that probably can be safely given in normal therapeutic doses to
G6PD-deficient patients without haemolytic anaemia
Acetaminophen (paracetamol, Tylenol, Tralgon,
hydroxyacetanilide)
Acetophenetidin (phenacetin)
Acetylsalicylic acid (aspirin)
Aminopyrine (Pyramidon, aminopyrine)
Antazoline (Antistine)
Antipyrine
Ascorbic acid (vitamin C)
Benzhexol (Artane)
Chloramphenicol
Chlorguanidine (Proguanil, Paludrine)
Chloroquine
Colchicine
Diphenyldramine (Benadryl)
Isoniazid
L-Dopa
Menadione sodium bisulfite (Hykinone)
p-Aminobenzoic acid
p-Aminosalicylic acid
Phenylbutazone
Phenytoin
Probenecid (Benemid)
Procainamide hydrochloride (Pronestyl)
Pyrimethamine (Daraprim)
Quinine
Streptomycin
Sulfacytine
Sulfadiazine
Sulfaguanidine
Sulfamerazine
Sulfamethoxazole (Gantanol)
Sulfamethoxypyridazine (Kynex)
Sulfisoxazole (Gantrisin)
Tiaprofenic acid
Trimethoprim
Tripelennamine (Pyribenzamine)
Vitamin K
Beutler E. Glucose-6-phosphate dehydrogenase deficiency: A historical perspective. Blood 2008; 111: 16-24
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CHAPTER 17 • Red blood cell enzyme defects
transfusion requirement, splenomegaly and gallstones at early age. Chronic haemolysis
attributable to G6PD deficiency is sometimes exacerbated by co-inherited (and unrelated)
genetic erythrocyte alterations, such as membrane defects, thalassaemia, glucose6-phosphate isomerase deficiency, pyruvate kinase deficiency and congenital
dyserythropoietic anaemia. Unexpectedly high amounts of non-conjugated bilirubin
can be seen in the co-inheritance of G6PD deficiency and Gilbert’s syndrome (8).
2.1.2 Epidemiology and molecular studies
The highest frequencies of G6PD deficiency have a global distribution that is very similar
to that of malaria (Africa, the Mediterranean region, Middle East and sub tropical Asia),
lending support to the so-called malaria protection hypothesis. However as a result
of migration, the disorder is also found in North and South America and in northern
European countries. In 1967, an expert committee of the WHO proposed standard
biochemical procedures for characterising variants, such as enzyme activity, Km for
glucose 6-phosphate, heat stability and electrophoretic pattern (10). After this, the
WHO introduced a classification of G6PD variants into five classes according to the
severity of the clinical phenotype (Table 3). Class I: severe enzyme deficiency with chronic
nonspherocytic haemolytic anaemia; Class II: severe enzyme deficiency (less than 10%
of normal); Class III: moderate-to-mild enzyme deficiency (10%-60% of normal);
Class IV: mild or no enzyme deficiency (60%-100% of normal); and Class V: increased
enzyme activity more than twice normal. There appeared to be 2 types of mutations
among Africans: G6PD A, a normally active enzyme with rapid electrophoretic mobility,
and G6PD A–, an enzyme with the same mobility as G6PD A, but with diminished activity.
The enzyme variant found in Mediterranean subjects was designated as G6PD B– but
Table 3: WHO classification of G6PD deficient variants
CLASS I
Severe G6PD deficiency (< 1%) associated with chronic haemolytic anaemia (CHA)
• Very rare variants (sporadic)
CLASS II
Severe G6PD deficiency (1-5%) associated with acute haemolytic anaemia (AHA)
• G6PD Mediterranean variants (polymorphic)
• G6PD Asiatic variants (polymorphic)
CLASS III
Moderate to severe G6PD deficiency (5-20%) associated with AHA
• G6PD A-
CLASS IV
Normal G6PD activity (100%)
• G6PD B+
• G6PD A+
CLASS V
Hyperactive G6PD activity (> 150%)
• G6PD Hecktoen
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subsequently renamed G6PD Mediterranean. G6PD Mediterranean variant is by no
means the only mutant enzyme found in the Mediterranean region; other enzyme variants
such as G6PD Seattle are common as well. In Asia even more heterogeneity was found.
These standard methods of WHO for differentiating variants from one another were used
by most investigators and until 1990, about 450 apparently different variants were
described. These methods, however, were not precise, and there was obviously a
professional advantage for an investigator to describe a new variant rather than
rediscover an old one. Thus, minor differences, due to technical variation, may be the
basis of describing the same variant under several different names. In this situation,
it has become increasingly difficult to decide whether a new variant actually differs
from all those that had been described before.
After isolation of G6PD cDNA, the primary structure of the enzyme was determined,
allowing the understanding of the polymorphisms of G6PD gene in a much more
precise fashion than had been possible when differentiation of variants depended on
enzyme kinetics, heat stability, and electrophoretic mobility. Accordingly, it was
possible to show quite rapidly that Yoshida’s deduction that G6PD A was a result of a
substitution of aspartic acid for asparagine had been quite correct. Interestingly, it
became apparent that G6PD A– is not a single variant but a group of variants that have
in common the same mutation as G6PD A+, together with one additional mutation,
usually c.202 GA (V68M) (11). The current database of some 160 mutants so far studied
indicate that G6PD gene mutations are due, with a few exceptions, to single missense
point mutations, entailing single amino acid replacements in the G6PD protein. The
exceptions are small deletions (of one to eight amino acids) found in very few
individuals. In most cases these mutations cause G6PD deficiency by decreasing the
in vivo stability of the protein producing a great acceleration of G6PD activity decrease
with red cell aging. In some variants the amino acid replacement can also affect the
catalytic function of the enzyme (for instance, a decreased affinity of G6PD for the
substrate) leading to a much more severe clinical phenotype such as chronic haemolytic
anaemia even in the absence of any oxidant challenge (WHO Class 1 variants). Recently
it has been demonstrated that the cluster of mutations around exons 10 and 11
designates the subunit interface, which interacts with other important residues located
elsewhere but which is brought close to this domain by protein folding. Stability of
the active quaternary structure is crucial for normal G6PD activity (11).
2.2 Pyruvate kinase (PK) deficiency
PK catalyzes the irreversible transfer of a phosphoryl group from phosphoenolpyruvate
(PEP) to ADP, thus yielding pyruvate and ATP; it is a regulatory key enzyme of the
glycolytic pathway (Figure 1). There are four isozymes of PK: PK-M1, PK-M2, PK-L, and
PK-R. The PK-M1 isoenzyme is expressed in skeletal muscle, heart, and brain, and it
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CHAPTER 17 • Red blood cell enzyme defects
is the only isoenzyme that is not subject to allosterically regulation. The PK-M2
isenozyme is expressed in early foetal tissues, but also in most adult tissues, including
leucocytes and platelets. PK-R is exclusively expressed in RBCs, whereas PK-L is
predominantly expressed in the liver (12). The PK-R and PK-L subunits are both
transcribed from the single gene PK-LR by the use of alternative promoters (Figure 4).
The active PK-R enzyme is a tetramer of four identical subunits and each subunit is
divided into 4 domains (Figure 5). The enzyme is allosterically activated by its
Figure 4: Genetic mechanism of liver PK (PK-L) and red blood cell PK (PK-R)
isozymes synthesis by selective transcription from a single gene PK-LR using
alternative promoters
PK-R ISOZYME (RBCs)
1
3
4
5 6
7
8
9 10
11
12
3
5'
2
3
4
5
6
7
8
9 10
11
12
PK-L ISOZYME (Liver)
NO CODING SEQUENCE
EXONS
CODING SEQUENCE
INTRONS
Figure 5: PK-LR monomer. Molecular model based on cat muscle PK enzyme
B DOMAIN
Active Centre
A DOMAIN
FDP Binding site
N DOMAIN
C DOMAIN
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substrate fructose-bis-phosphate (FBP) and negatively regulated by ATP. Furthermore,
PK has an absolute requirement for cations Mg2+ and Ca+.
2.2.1 Clinical manifestations
PK deficiency (OMIM 266 200) is the most common enzymopathy associated with CHA,
and about 300 patients have been reported so far. CHA usually occurs in compound
heterozygotes for two different mutant alleles and in homozygotes, where the
increased 2,3-BPG levels ameliorate the anaemia by lowering the oxygen-affinity of
haemoglobin. Phenotypically, the clinical picture varies from severe haemolysis
causing neonatal death, to a well-compensated haemolytic anaemia and only very
rare cases can present with hydrops foetalis.
2.2.2 Epidemiology and molecular studies
The estimated prevalence of PK deficiency is 51 cases (i.e., homozygous or compound
heterozygous patients) per million in the white population. In 1979, recommended
procedures for the biochemical characterisation of PK variants were issued by a
subcommittee of the International Committee for Standardisation in Haematology
(13). More recently mutations of PK gene leading to CHA have been extensively
studied using molecular biology procedures. By the year 2008, more than 180 gene
mutations in PKLR had been reported to be associated with PK deficiency (14). Most
of these mutations (70%) are the missense mutants c.1456CÆT (Arg486Trp),
c.1529GÆA (Arg510Gln), c.994GÆA (Gly332Ser), and the nonsense mutant
c.721GÆT (Glu241stop). They affect conserved residues in structurally and
functionally important domains of PK. A clear geographical distribution has been
observed among the most frequent mutations of PK enzyme (Table 4).
2.3 Glucose phosphate isomerase (GPI) deficiency
GPI deficiency (OMIM 172 400) is the second most common erythroenzymopathy of
Table 4: Geographical distribution of the most frequent PK-LR gene mutations
leading to chronic haemolytic anaemia (CHA)
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Central Europe and North-America
G1529 A (Arg510Glu)
C1594 T (Arg 532Trp)
G 994 A (Gly332Ser)
Southern Europe
C1456T (Arg486Trp)
Fatal mutations (CHA in heterozygotes)
G409A (Ala137Thr) (South Europe)
G1091A (Gly364Asp) (Northern Europe)
G721T (Glu241stop) (Worldwide)
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CHAPTER 17 • Red blood cell enzyme defects
glycolytic enzymes after PK deficiency, and approximately 50 different cases have been
described to date. GPI deficiency is an autosomal recessive genetic disorder associated
with mild to severe CHA in homozygotes or compound heterozygotes. In a very few
cases, GPI deficiency is associated with neurological impairment and granulocyte
dysfunction. Twenty-nine mutations have been detected in GPI, 24 of which were
missense mutations, 3 were nonsense mutations, and 2 were mutations that affected
splice sites. Mapping of these mutations to the crystal structure of human GPI has
provided insight into the molecular mechanisms causing haemolytic anaemia in this
disorder. In accordance with the 3-dimensional structure, mutations can be categorised
into 3 distinct groups that affect the overall structure, the dimer interface, and the
active site. Nearly all GPI mutants described are heat unstable, whereas kinetic
properties are more or less unaffected (15).
2.4 Pyrimidine 5’ nucleotidase (P5’N-1) deficiency
P5’N-1, uridine monophosphate hydrolase-1 (UMPH-1) or cytosolic 5:nucleosidase
II (cN-III) is an enzyme which major role is in the catabolism of the pyrimidine
nucleotides, uridine monophosphate (UMP) and cytidine monophosphate (CMP),
mainly resulting from RNA degradation during erythrocyte maturation.
P5’N-1 deficiency (OMIM 606 224) is an autosomal recessive disorder characterised by
CHA with marked reticulocytosis and increased concentrations of pyrimidine nucleotides
within mature erythrocytes A characteristic RBC morphological abnormality is a heavy
basophilic stippling, and its observation is very helpful for P5’N diagnosis (Figure 6).
Since the first description of P5’N in 1974, about one hundred P5’N-deficient patients
have been reported worldwide
Figure 6: Severe basophilic stipling
although it is possible that a larger
spontaneous aggregation of ribosomal
number have been not detected. It
RNA in the cytoplasm of erythrocytes
has been suggested that P5’N-1
in a case of P5’N-1 deficiency
deficiency is the third most common
RBC enzymopathy, after G6PD and PK
deficiencies, although this is not the
case for the different geographical
areas (16). P5’N-1 deficiency can also
be acquired as a result of lead
poisoning or oxidative stress. Lead is
a powerful inhibitor of P5’N and
determination of lead levels should
be included whenever the constellation
of haemolytic anaemia, P5’N
deficiency, and basophilic stippling is
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found. Lead-induced acquired P5’N deficiency is treatable, unlike the congenital
deficiency for which no therapy is available (16). It is worth mentioning several
clinical reports of severe haemolytic anaemia in patients with co-inheritance of
P5’N deficiency and the thalassaemic haemoglobin Hb E. This provides a further
support to the idea that P5’N is particularly susceptible to oxidative damage resulting
from the instability of haemoglobin in thalassaemic syndromes (17).
2.5 Enzymopathies associated with haemolytic anaemia and systemic disease
Some very rare enzymopathies, in addition to causing acute or chronic haemolytic
anaemia, are associated with systemic (non-haematological) manifestations such
as neuropathy with or without mental retardation, myopathy, recurrent infections
and metabolic acidosis (Table 5). Understanding of the clinical behaviour of these
enzymopathies requires a deep knowledge of their genetics, biochemical properties
and the structural consequences of responsible mutations on the enzyme molecule.
This phenotype, however, is not only depending on the molecular properties of
mutant proteins but also on a complex interplay of physiologic, environmental, and
other genetic factors (polymorphisms posttranslational modifications, epigenetic
modification, etc). Moreover, aberrant enzymatic function in non-erythroid tissues
may also influence the clinical outcome of hereditary enzymopathies. Their study
is also fostered by the growing recognition that several glycolytic enzymes have
diverse, non-enzymatic functions (18), including modification of cell motility, control
Table 5: Erythroenzymopathies associated with haemolytic anaemia and systemic
disease
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1
Phosphofructokinase (PFK)deficiency
- Muscle disease (glycogenosis Type VII)
(OMIM 171 850)
2
Triose Phosphate Isomerase (TPI) deficiency
- Progressive neurologic disorder (Spasticity)
- Recurrent infections
(OMIM 190 450)
3
Phosphoglycerate kinase (PGK) deficiency
- Neuromuscular manifestations (rhabdomyolysis)
(OMIM 300 653)
4
Adenylate kinase deficiency (AK) deficiency
- Psychomotor impairment (mental retardation)
(OMIM 103 000)
5
Glutathione synthetase (GS) deficiency
(OMIM 266 130)
- Metabolic acidosis (5-oxoprolinuria)
- Neurological symptoms (psychomotor retardation and ataxia)
- Recurrent bacterial infections
6
Gamma-glutamylcysteine synthetase (GGS) deficiency
- Metabolic acidosis (5-oxoprolinuria)
- Neurological symptoms (psychomotor retardation and ataxia)
- Recurrent bacterial infections
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(OMIM 230 540)
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of apoptosis and regulation of oncogenes. In addition these have important roles
in the regulation of the transcription of an array of genes by multiple mechanisms.
Accordingly it remains to be determined whether the non-erythroid clinical
phenotypes of mutations of some red cell enzyme genes can be explained by their
newly discovered non-glycolytic roles. Hence, future research in RBC enzymopathies
should take all these new factors and phenotypic modifiers into account when
considering the relationship between genotype and phenotype correlation.
3. Erythroenzymopathies associated with methaemoglobinaemia
Hereditary methaemoglobinaemia can be divided into two forms. One is due to Hb
M, which has autosomal dominant inheritance, and the other is due to an enzyme
abnormality of the NADH-dependent methaemoglobin reductase system. The later
has an autosomal recessive inheritance and is due to cytochrome b5 reductase (b5R)
deficiency (19). The two disorders can be diagnosed by the absorption spectrum
of a clear, stroma-free haemolysate.
Methaemoglobinaemia due to b5R deficiency (OMIM 250 800) can be classified into
two main groups with drastically different clinical manifestations. In one group,
patients not unwell and the only clinical manifestation is cyanosis (hereditary b5R
deficiency type I), which can be treated with the administration of methylene blue.
In the other group, in addition to cyanosis there is a much more severe clinical
syndrome characterised by microcephaly, opisthotonos, retarded growth and
progressive neurological impairement leading to generalysed hypertonia, mental
retardation and death before puberty (hereditary b5R deficiency type II).
Unfortunatelly this clinical form of hereditary b5R deficiency has no treatment but
it is very rare.
3.1 Hereditary b5R deficiency type I
This is found worldwide, but it is endemic in some populations such as the
Athabascan Indians, Navajo Indians, and Yakutsk natives of Siberia. In other ethnic
and racial groups, the defect occurs sporadically. Homozygotes or compound
heterozygotes have methaemoglobin concentrations of 10 to 35% and appear
cyanotic but are usually asymptomatic even with levels up to 40%. Life expectancy
is not shortened, and pregnancies occur normally. Significant compensatory elevation
of haemoglobin concentration is sometimes observed (erythrocytosis). The b5R
activity of the erythrocytes of heterozygotes is approximately 50% of normal.
Although this activity level is sufficient to maintain normal methaemoglobin levels
during normal conditions, oxidant stress can overwhelm the erythrocyte’s capacity
to reduce methaemoglobin and produce acute symptomatic methaemoglobinaemia.
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The cyanosis can be effectively treated with methylene blue or ascorbic acid and
it is only indicated for cosmetic reasons, because it has no effect on the neurological
abnormalities.
3.2 Hereditary b5R deficiency type II
This is less frequent than the former, and patient’s life expectancy is significantly
shortened due to the neurological complications which are very severe. Enzyme
deficiency is generalised and manifested by a nearly total lack of microsomal
cytochrome b5 reductase in all the cells. Amniotic cells contain easily measurable
b5R activity; thus, prenatal diagnosis of homozygous b5R deficiency is feasible.
From molecular point of view, in hereditary b5R deficiency type I the enzyme
deficiency is limited to the RBCs and the abnormal gene product is produced at a
normal rate but is unstable. As a result in b5R deficiency type I, only mature RBCs,
which cannot synthesise proteins, are affected. By contrast in b5R deficiency type
II, mutations cause underproduction of the enzyme or an enzyme with decreased
enzymatic activity with a generalised enzyme deficiency in all cell types.
In summary, congenital methaemoglobinaemias, with the exception of type II
congenital methaemoglobinaemias, are asymptomatic and only of cosmetic concern
to some. In contrast, a comparable level of methaemoglobin in patients with
acquired methaemoglobinaemia is symptomatic due to severe acute tissue hypoxia
which may be life-threatening.
4. Erythroenzymopathies associated with erythrocytosis
Congenital erythrocytosis can result from a extremely rare RBC enzymopathy due
to bisphosphoglycerate mutase (BPGM) deficiency (OMIM 222 800). Only two
affected families have been described so far. In both cases patients had a complete
deficiency of erythrocyte BPGM, increased ATP levels, ruddy cyanosis, high
haemoglobin concentration (190g/L) and no evidence of haemolysis (20). BPGM is
a tri-functional enzyme whose main function is to synthesise 2,3-diphosphogycerate
(2,3-BPG). In the erythrocyte, 2,3-BPG is synthesised and dephosphorylated in a
special bypass called “Rapoport-Luebering shunt” (Figure 1). This glycolytic bypass
is unique to mammalian red blood cells and represents an important physiologic
means for regulating the oxygen affinity of haemoglobin, which is also influenced
by changes of blood pH. 2,3-BPG is synthesised by BPGM, an enzyme that also
displays two other activities, a phosphatase activity that degrades 2,3-BPG and a
minor mutase activity identical to that of glycolytic phosphoglycerate mutase.
Quantitatively, 2,3-BPG is the major glycolytic intermediate. Increased 2,3-BPG levels
result in a decrease of affinity of haemoglobin for oxygenwhich is more readily
transferred to tissue. The anaemia is thus ameliorated and exercise tolerance is
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improved. This beneficial effect is absent in the distal glycolytic enzyme defects
HK, GPI, PFK, aldolase, and TPI deficiency that all cause a decrease in 2,3-BPG levels.
Several of the distal glycolytic enzymes (i.e., HK and PFK) are inhibited by 2,3-BPG.
References
1. Kahn A. Abnormalities of erythrocyte enzymes in dyserythropoiesis and malignancies. Clin
Haematol 1981; 10: 123-138.
2. Tanaka KR, Paglia DE. Pyruvate kinase and other enzymopathies of the erythrocyte. In:
Scriver CR, Beaudet AL, Sly WS, Valle D (eds): The Metabolic and Molecular Basis of
Inherited Disease (ed 7). New York, NY, McGraw-Hill, 1995, p. 3485.
3. Eber SW. Disorders of erythrocyte glycolysis and nucleotide metabolism. In: Handin RI,
Lux SE, Stossel TP, eds. Blood Principles and Practice of Hematology. 2nd ed. Philadelphia,
PA: Lippincott Williams & Wilkins; 2003: 1887-1914.
4. Prchal JT, Gregg XT. Red cell enzymopathies. In: Hoffman R, Benz E, eds. Hematology:
Basic Principles and Practice, 4th ed. 2005: 653–659.
5. Beutler E. Red Cell Metabolism. A Manual of Biochemical Methods. 3rd Ed. Orlando, FL,
Grune & Stratton, 1984.
6. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Haematology. 10th Ed. Curchill
Livingstone, 2006.
7. Naylor CE, Rowland P, Basak K, et al. Glucose 6-phosphate dehydrogenase mutations causing
enzyme deficiency in a model of the tertiary structure of the human enzyme. Blood 1996;
87: 2974-2982.
8. Luzzatto L, Metha A, Vulliamy T. Glucose 6-phosphate dehydrogenase deficiency. In: Scriver
CR, Beaudet AL, Sly WS, et al, eds. The metabolic and molecular basis of inherited disease,
8th ed. Columbus: McGraw- Hill, 2001: 4517-4553.
9. Kaplan M, Hammerman C. Glucose-6-phosphate dehydrogenase deficiency: A hidden risk
for kernicterus. Semin Perinatol 2004; 28: 356–364.
10. Betke K, Beutler E, Brewer GJ, et al. Standardization of procedures for the study of glucose6-phosphate dehydrogenase. Report of a WHO scientific group. WHO Tech Rep Ser No.
366, 1967.
11. Hirono A, Beutler E. Molecular cloning and nucleotide sequence of cDNA for human
glucose-6-phosphate dehydrogenase variant A(–). Proc Natl Acad Sci USA 1988; 85:
3951–3954.
12. Kanno H, Fujii H, Miwa S. Structural analysis of human pyruvate kinase L-gene and
identification of the promoter activity in erythroid cells. Biochem Biophys Res Commun
1992; 188: 516-523.
13. Miwa S, Boivin P, Blume KG, et al. Recommended methods for the characterization of red
cell pyruvate kinase variants. Br J Haematol 1979; 43: 275.
14. Zanella A, Fermo E, Bianchi P, et al. Pyruvate kinase deficiency: The genotype-phenotype
association. Blood Rev 2007; 21: 217-231.
15. Repiso A, Oliva B, Vives Corrons JL, et al. Red cell glucose phosphate isomerase (GPI):
A molecular study of three novel mutations associated with hereditary nonspherocytic
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hemolytic anemia. Hum Mut 2006, #937, online.
16. Vives-Corrons JL. Chronic non-spherocytic haemolytic anaemia due to congenital
pyrimidine 5’nucleotidase deficiency: 25 years later, Bailliere’s Best Pract Res Clin
Haematol 2000; 13: 103–118.
17. Zanella A, Bianchi P, Fermo E, Valentini G. Hereditary pyrimidine 5‘-nucleotidase
deficiency: From genetics to clinical manifestations. Br J Haematol 2006; 133: 113–123.
18. Kim J, Dang CV. Multifaceted roles of glycolytic enzymes. Trends Biochem Sci 2005; 30:
142–150.
19. Jaffe ER, Hultquist DE. Cytochrome b5 reductase defi ciency and enzymopenic hereditary
methemoglobinemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds), The Metabolic and
Molecular Basis of Inherited Disease (ed 7), New York, NY,McGraw-Hill, 1995, p. 3399.
20. Hoyer JD, Allen SL, Beutler E, et al. Erythrocytosis due to bisphosphoglycerate mutase
deficiency with concurrent glucose-6-phosphate dehydrogenase (G-6-PD) deficiency.
Am J Hematol 2004; 75: 205-208.
Multiple Choice Questionnaire
To find the correct answer, go to http://www.esh.org/iron-handbook2009answers.htm
1. The most frequent RBC enzymopathy associated with chronic
haemolytic anaemia is:
a) Pyruvate kinase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b) Glucose-6-phosphate dehydrogenase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c) Pyrimidine 5' nucleotidase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d) Reduced glutathione deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Acute haemolysis after fava beans ingestion is a usual clinical
manifestation of:
a) Glutathione synthetase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b) Pyruvate kinase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c) Glutathione reductase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d) Pyrimidine 5' nucleotidase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Diagnosis of pyruvate kinase deficiency requires the measurement
of enzyme activity on:
a) Whole blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b) Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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c) Leukocyte-free haemolysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d) Blood serum or plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. One of the following RBC enzymopathies can be associated with
progressive neuropathy:
a) Glucose-6-phosphate dehydrogenase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b) Glucose-phosphate isomerase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c) Triose phosphate isomerase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d) Aldolase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Basophilic stippling is a RBC abnormality that can be frequently
present in:
a) Oxidative metabolism impairment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b) Increased reduced glutathione (GSH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c) Adenosine deaminase (ADA) hyperactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d) Pyrimidine 5' nucleotidase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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